Cydia pomonella (Linnaeus, 1758)

Kuyulub, Akın, Genç, Hanife, Online, Published & Version, Final, 2020, Genetic diversity of codling moth Cydia pomonella L. (Lepidoptera: Tortricidae) populations in Turkey, Turkish Journal of Zoology 44 (5), pp. 462-471 : 464-466

3.1. Genetic diversity in C. pomonella populations Mitochondrial COI is known to be a suitable tool to study genetic structure and genetic distance among pest species

( Li et al., 2015). A total of 61 individuals representing 9 Turkish population of C. pomonella yielded about 708 bp of mitochondrial fragments, and these were successfully sequenced. To eliminate sequencing artifact errors, we used only 631 bp of COI fragments by trimming the beginning and end of the sequences. All sequences corresponded to bases 1525-2055 of the C. pomonella COI region in the complete mitochondrial genome ( Shi et al., 2013). Neither insertion/deletion nor stop codon/nonsense mutations were observed. The neighbor-joining model of the COI sequences using the Kimura 2-parameter showed 2 distinct groups: one consisted of 9 individuals in the Çanakkale and Balıkesir populations, while the other consisted of 52 individuals in all tested populations divided by a node with 96% accuracy ( Figure 1 ).

The sequences of 631 bp of the COI gene region contained 604 constant sites and 27 polymorphic characters, 13 of which were singletons and 14 of which were parsimony informative. The average base frequency was A: 29.63%, T: 39.86%, C: 15.93%, and G: 14.59%, indicating a strong AT bias (69.5%). AT-rich nucleotide compositions are also commonly known in COI gene analyses ( Zhang and Hewitt, 1996). Mitochondrial DNAlike sequences (numts) were not observed. Twenty-one substitutions occurred at the 3rd codon position (77.8%), 2 substitutions were observed at the 2nd codon position (7.4%), and 4 substitutions were at the 1st codon position (14.8%). The transition/transversion (Ts/Tv) ratios were k 1 = 84.477 (purines) and k 2 = 157.941 (pyrimidines). A transversion was observed at site 367 and 27 transitions (in all other substitutions sites), mostly being C/T (49.4), followed by T/C (19.73). The overall transition/ transversion bias was R = 55.452 ( Tamura et al., 2004). The average number of nucleotide differences was k = 3.600.

From all 61 sequenced samples, we observed a total of 22 mitochondrial COI haplotypes (Table 2). When the haplotypes were examined, 17 haplotypes were private (unique to a single population), and 5 haplotypes were shared between populations (Table 2). Haplotype H 2 (23/61) was the 1st frequently shared haplotype and was evident in 7 populations/localities (Çanakkale, Ordu, Bursa, Balıkesir, İzmir, İstanbul, and Adana). Haplotype H 3 (8/61), the 2nd most frequently shared haplotype, was found in 3 populations/localities (Çanakkale, Bursa, and Balıkesir) (Table 2). One COI haplotype (H1) was distributed by populations from Çanakkale, and Balıkesir. Nine COI gene haplotypes (H4, H5, H6, H7, H8, H9, H10, H11, and H12) were only distributed by populations from Çanakkale. One COI haplotype (H13) was distributed by populations from Balıkesir, and only 2 COI gene haplotypes (H14 and H15) were distributed by populations from Bursa. One COI haplotype (H16) was distributed by populations from Bursa and Erzurum, and

KUYULU and GENÇ / Turk J Zool

6 COI haplotypes (H17, H18, H19, H20, H21, and H22) were distributed by populations from other provinces but were not shown in Çanakkale (Table 2).

The genetic diversity of C. pomonella populations showed that haplotype variation ranged from 0.00 to 1.000 ± 0.500 with a mean haplotype diversity of 0.835 ± 0.041 ( Table 3). The highest haplotype variation was observed in the Balıkesir, İstanbul, Erzurum, Adana, and Van populations, followed by the Bursa population (h = 0.933 ± 0.122). The Ordu population showed no haplotype variation because of the detection of only a single type of haplotype in this population ( Table 3). However, nucleotide diversity varied between 0.00% and 1.162% ± 0.414%. The mean nucleotide variation was estimated as π = 0.571% ± 0.094%. The Balıkesir population showed the highest nucleotide diversity (1.162% ± 0.414%), followed by the Çanakkale population (0.708% ± 0.123%) ( Table 3). The mismatch distribution showed the multimodal profile. The Harpending raggedness index was low (r = 0.0441). The results of neutrality tests showed that Tajima’s D = –1.280, P> 0.10, and Fu’s Fs = –9.021, P <0.001 .

A pairwise matrix among C. pomonella haplotypes was used to show the sequence differences. The p-distances model among haplotypes ranged from 0.16% to 2.57% (1 to 16 bp), respectively ( Table 4). A nucleotide difference was monitored between 18 cases, while the highest nucleotide difference (16 bp) was detected between H6 (from Ayvacık and Çanakkale) and H5 (from Ezine and Çanakkale) ( Table 4).

The pairwise Fst calculations indicated a small amount of genetic differentiation, (Table 5) particularly in 2 locations and indicated a little differentiation from some other populations; the Çanakkale population differed from the Adana, Erzurum, and Ordu populations (Fst> 0.1). Erzurum was different from Ordu and Adana (Fst> 0.1). Other populations showed genetic differentiation values on a scale of 0 and 0.1 (Table 5).

3.2. Phylogenetic relationships among codling moth haplotypes

The estimation of phylogenetic relationships was conducted on 22 C. pomonella mitochondrial haplotypes. The maximum likelihood (ML) tree of the COI gene T able 2. Mithochondrial haplotypes distribution of codling moth among different populations.

haplotypes is shown in Figure 2 . The ML tree produced 2 well-supported clades by high bootstrap values. Clade I is composed of basally-located 3 haplotypes (H6, H1, and H11), and H8 from the Çanakkale and Balıkesir populations is referred to as “Western-Turkey Populations.” On the other hand, Clade II is made of basally-located haplotypes (H14) from Bursa population. All other haplotypes bifurcated into small subclades. Therefore, Clade II haplotypes are referred to as “invasive and adaptive populations.’’

3.3. Haplotype network of the mitochondrial COI gene MJ network analysis among the COI haplotypes produced similar results to those in the ML tree. The MJ network tree suggested that Turkish haplotypes of C. pomonella were clearly divided into 2 major clades from each other by at least 4 mutation steps ( Figure 3 ). The common H1 (from Çanakkale and Balıkesir) haplotype was connected to H6 (from Ayvacık and Çanakkale) and H11 (from Umurbey- Lapseki and Çanakkale) in Clade I. H6 was also found as the most distant haplotype with 4 mutation steps connected to H1. The H8 (Clade I) haplotype (from Center and Çanakkale) was connected to a unsampled intermediate haplotype by 4 mutation steps. In Clade II, H2 (N = 23), the most common haplotype, was shared among all tested populations of C. pomonella , excluding the Balıkesir and Erzurum populations. In addition, H2 was connected to several haplotypes including the 8 haplotypes H3, H9, H12, H14, H16, H17, H20, and H21, which is the one unsampled haplotype. It is remarkable that H3 (from the Çanakkale, Bursa, and Balıkesir populations) (N = 8) was connected to 6 haplotypes (H2, H5, H7, H15, H19, and H22) and that H9 (from the Çanakkale populations) (N = 3) was connected to the following 4 haplotypes: H2, H10, H15, and H18—and to 2 unsampled haplotypes ( Figure 3 ).

Figures

Figure 1. Relationships among C.pomonella Turkish individuals (the red label shows the 1st group and the black label shows the 2nd group).

Figure 2. Maximum likelihood tree of the COI gene haplotypes of C. pomonella. Numbers above the branches indicate the bootstrap values (>50) for nodes (1000 replications).

Figure 3. Median Joining Network based on mitochondrial COI haplotypes. Each circle shows a haplotype, and the circles are related with observed specimens. Colors within the nodes indicate C. pomonella sampling areas. Turkish populations of C. pomonella show the 2 clades.